The invention relates to a method and a reagent for the photometric determination of the activity of creatine phosphokinase (E.C. 2.7.3.2), hereinafter called CK, by means of the bioluminescent system of fireflies.
CK catalyzes the following reaction: ##STR1##
In the human body, two different kinds of sub-units of this enzyme occur, the sub-units M and B. Since the active enzyme is composed of two sub-units, and since the two sub-units can combine freely with one another, three types of enzyme are possible, the muscle type (CK-MM), the brain type (CK-BB) and the hybrid type (CK-MB), which occurs almost exclusively in the myocardium and enters into the serum in myocardial infarction, where it can be measured in an increased concentration. The activity of this isoenzyme, in addition to the total activity of CK in the serum, is of great importance in the diagnosis of myocardial infarction.
CK activity is commonly determined by an absorption photometry method in which the formation of ATP (cf. Equation 1) is measured in the following manner: ##STR2##
This determination of the CK activity by measuring the amount of ATP that has been formed is interfered with by the adenylate kinases (E.C. 2.7.4.3 "myokinases") of the serum (originating from the liver, muscles or erythrocytes), which catalyze the following reaction: ##STR3##
Their activity can be most effectively inhibited by the addition of 5 to 10 millimoles per liter of adenosine-5'-monophosphate (AMP) and 10 micromoles per liter of diadenosine pentaphosphate (Ap5A), which eliminates their interference with the absorption photometry test. The interference can also be eliminated by using 1 to 10 millimoles per liter of NaF instead of Ap5A. An important disadvantage of these methods is a lag phase which cannot be reduced to less than 90 seconds. Another important disadvantage of the absorption photometry method lies in its poor sensitivity, which permits the measurement only of activities above 10 units per liter, and therefore makes it virtually impossible to measure the CK-MB activity, particularly in the low pathological range, as well as in the normal range.
Consequently a number of attempts have been made to detect the formation of ATP by CK through the luciferase bioluminescence of the firefly. This method has the advantage of greater sensitivity and the absence of the lag phase.
The luciferase of the firefly (Photinus pyralis et al.) catalyzes the following reaction: ##STR4##
The light that is produced in this reaction is emitted with a yield of virtually 1 Einstein per mole of ATP. It has a wavelength of 562 nm at the peak. The reaction is extremely sensitive, and permits the quantitative determination of ATP concentrations down to 10.sup.-13 moles per liter.
It is known, however, that the firefly Reaction 5 is so greatly inhibited by AMP (Arch. Biochem. Biophys. 141, 49, 1970) that the practical application of this reaction in the measurement of CK activities in accordance with Equation 1 in the serum is very problematical, since it prevents the use of AMP as an inhibitor of the adenylate kinase in Reaction 4 (Proc. Nat. Acad. Sci. 71, 1384 to 1387 (1974)). It is furthermore known to partially eliminate the necessity of the inhibition of adenylate kinase by performing the reaction (1) at a sub-optimal ADP concentration, that is, without substrate saturation, and thus operating at a substrate concentration at which, on account of the rather steep curve representing the activity of adenylate kinase in relation to the substrate concentration, the activity of the adenylate kinase is negligibly small in comparison to that of the CK. In this manner, the CK in the serum can be measured along with the adenylate kinase by the bioluminescent method (Clin. Chim. Acta 87, 199 to 209 (1978)), but, of course, considerable disadvantages must be accepted:
1. The CK reaction no longer is performed on the basis of a pseudo zero order. PA1 2. The recovered CK activity, with respect to an internal calibration with ATP, amounts to only about 11% of the values measured by the absorption photometry test. PA1 3. Slight inaccuracies in the ADP concentration have a very great effect on the results. PA1 1. The adenylate kinases can be completely inhibited, PA1 2. The activity measured by means of bioluminescence expressed in I.U. (micromoles of substrate transformation per minute) is equal to the activity measured in the mixture used in the absorption photometry, and PA1 3. The procedure can be performed with the CK saturated with the substrate.